Structures and mechanism of chitin synthase and its inhibition by antifungal drug Nikkomycin Z

, Chitin, one of the most common polysaccharides in nature, is produced by fungi, insects, ﬁ sh, etc. Chitin is a linear N-acetylglucosamine (GlcNAc) polymer with β -1,4-linkage and is synthesized by membrane-integrated chitin synthase (Chs). As the essential polysaccharide in cell walls of prominent fungal pathogens, chitin plays vital roles in cell wall assembly and cell integrity maintenance for fungi 1 . Chitin biosynthesis, therefore, becomes an appealing target for antifungal drug development 1,2 . One well-known example is Nikkomycin Z (NikZ), the competitive inhibitor of Chs 3,4 (Fig. 1a). NikZ is under clinical development for treating coccidioidomycosis, blas-tomycosis, and histoplasmosis 2,5,6 . However, the mechanism of action of Chs and its inhibition by NikZ remain enigmatic.


Supplementary Discussions
Our Chs1 structure provides new insights into the GT2 family of membrane-integrated processive GTs (previously structurally represented by cellulose synthases such as BcsA and CesA). We found that Chs1, the GT2 member, adopts a novel fold. The structural comparison between Chs1 and BcsA reveals their limited overall structural similarity, with resemblance mainly limited to their cytosolic GT domains (Fig. S11a). Within GT domain and at the membrane-cytoplasm interface, their characteristic motifs such as the 'ED motif', the 'QxxRW motif', the interfacial helix IF1-3 are well matched between the two structures, suggesting a conserved catalytic mechanism in synthesizing chitin and cellulose (Fig. S11b,c). In contrast, the transmembrane domains of Chs1 and BcsA vary dramatically in numbers of TM helices as well as the topologies (Fig. S11d, e), resulting in differently organized transmembrane tunnels above the active site and likely different modes of product translocations through membranes.
Our study reveals that Chs1 forms a compact dimer through the extensive contacts on membrane domain as well as through a domain-swapping of the cytosolic CL1 region.
This dimerization mode is consistent with that of the recently reported structure of Candida albicans Chs2 where they show quite similar conformations (with root-meansquare-deviation RMSD of 1.2-Å over 666 aligned residues) 1 . Consistently, oligomer was also observed during the purification of insect chitin synthase 2 . Therefore, it is conceivable that the oligomerization of chitin synthase could underlie the formation of chitin microfibrils ( Supplementary Fig. S9c). There are two basic polymorphic types of chitin microfibrils: α-form and β-form where individual chitin chains are organized in antiparallel and parallel manner, respectively. Though this complexity leaves the assembly mechanism enigmatic, it would be tempting to speculate on the basis of dimeric architecture of Chs. For β-chitin, the two polymer chains extended on individual protomer of Chs dimer may directly assemble in a parallel manner. For α-chitin, the polymer synthesized on one Chs protomer could fold on itself through intra-chain contacts to form a sheet with anti-parallel arrangement 3 ; this could be followed by the inter-chain packing of a similar sheet from the other Chs protomer, forming the network of chitin microfibrils

Protein expression and purification
The codon-optimized full-length cDNA of S. cerevisiae Chs1 (Uniprot ID P08004) was subcloned into pCAG vector with a C-terminal 3xFLAG tag. HEK 293F suspension cells (Invitrogen) were cultured in freestyle 293 medium at 37 °C under 5% CO2 in shaker. The cultured cell at a density of ~ 2 × 10 6 cells per milliliter were transiently transfected with expression plasmid and polyethylenimines (PEI). For 1-liter cell culture, approximately 1.4 mg of plasmid was pre-mixed with 6 mg of PEI in 50 ml of fresh medium for 15-30 minutes at room temperature before transfection. The transfected cells were cultured for 48 hours before harvest.
For protein purification, the harvested cells were lysed by sonication on ice in buffer: 25 mM HEPES pH 7.4, 150 mM NaCl, 2 mM MgCl2 and protease inhibitor cocktail (Roche).

Grid preparation and Cryo-EM data acquisition
For cryo-EM analysis, three-microliter aliquots of the purified Chs1 at the concentration of 5 mg/ml were applied to glow-discharged grids (Quantfoil R1.2/1.3 Au, 300 mesh).
Then the grids were blotted for 5 seconds with 100% humidity at 4 °C and were plungefrozen in liquid ethane using an Vitrobot Mark IV system (Thermo Fisher Scientific). For Nikkomycin Z (NikZ) treated sample, NikZ at final concentration of 1 mM was added to purified Chs1 (5 mg/ml). The mixture was incubated at 4 °C for 30 minutes before freezing the cryo-EM grids. Cryo-EM data were recorded on a K3 camera (Gatan) in a 300 kV FEI Titan Krios electron microscopy (FEI). The datasets were automatedly collected with EPU (FEI). Micrographs were recorded in counting mode at a nominal magnification of 130,000× with a physical pixel size of 0.92 Å. Defocus values varied from -1.0 μm to -3 μm. A total exposure of 1.3 seconds was dose-fractionated into 32 sub-frames, resulting in a total accumulated dose of 50 electrons per Å 2 .

Cryo-EM data processing
The recorded movies were motion-corrected using MotionCor2 5 . CTFFIND4 was then used to estimated Contrast transfer function (CTF) parameter for individual micrograph 6 .
Further data processing was carried out using RELION-3.0 7 and similar strategy was used for the dataset of apo-Chs1 and the dataset of Chs1 treated with Nikkoymcin Z. For each dataset, a set of ~1000 particles were manually picked, which were subjected to Relion 2D classification to generate the templates for autopicking. Initial 3D model was generated using Relion 3D initial model module. One or two rounds of 3D classification without the application of symmetry were performed to eliminate particles of poor quality.
Subsequently, the good particles were selected for one round of 3D classification with C2 symmetry. The 3D class with high-resolution structural features were combined for 3D refinement, polishing and postprocess. Finally, for apo-Chs1 dataset, a map at an overall resolution of 3.2 Å was obtained from 93641 particles; for Chs1+Nikkomycin Z dataset, a map at an overall resolution of 2.9 Å was obtained from 188337 particles. The resolution was estimated using the gold standard Fourier shell correlation 0.143 criterion 8 . The distribution of local resolution distribution was estimated using ResMap 9 .

Model building and refinement
The initial model of Chs1 was obtained from the map of Chs1 complexed with Nikkomycin Z, generated de novo with the map_to_model in PHENIX 10 . It was further improved by manual adjustments and rebuilding in COOT 11 . The building process was aided by the good structural features around secondary structures, transmembrane helices as well as bulky residues. Nikkomycin Z and bound lipids were added according their densities. The refinement of Chs1+Nikkomycin Z model was carried out using real_space_refine module in PHENIX 10 . To build the apo-Chs1 model, the initial model was obtained from the Chs1+Nikkomycin Z model. It was rigid-body docked into the apo-Chs1 map and was further manually inspected and adjusted in COOT 11 . Then the apo-Chs1 model was refined using PHENIX 10 . The final models were assessed using MOLPROBITY 12 . Chimera, ChimeraX and PyMOL (Schrödinger, LLC.) were used to prepare the figures 13,14 . Statistics of the 3D reconstruction and model refinement were provided in Supplementary Table S1. EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) with accession codes EMD-33422 and EMD-33423. Atomic coordinates have been deposited in the Protein Data Bank (PDB) with accession codes PDB 7XS6 and 7XS7.

Chs1 activity assay
Site-directed mutagenesis was done with PCR overlapping extension and confirmed by sequencing. The Chs1 variants was purified as described above. The Chs1 activity was measured by monitoring the UDP generated, using UDP-Glo Glycosyltransferase Assay kit (Promega). Chs1 variants (2 μg) was added to 20 μL reaction mixture in total containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM MgCl2 and 0.04% GDN. The reaction was initiated by adding UDP-GlcNAc to a final concentration of 4 mM. Reactions were carried out at 30 °C for 1 hour. Luminescence was recorded using a Pherastar FS system (BMG Labtech). In the case of inhibitor profiling, serial dilutions of Nikkomycin Z drug were first incubated with Chs1 for 5 minutes at room temperature before adding other components to react.

Calcofluor White Staining
The synthesized chitin polymer was visualized by the fluorescent dye Calcofluor White 15 .
Wild-type or mutant Chs1 samples (2 μg) were added to a 20 μL reaction mixture   three interfacial helices IF1-IF3 and the broken TM1 are similarly organized to form the path connecting the membrane tunnel for chitin translocation, as indicated by the orange circle.
c, GT domains. Two interfacial helices IF1 and IF2, two characteristic motifs 'ED motif' and 'QxxRW motif' are indicated as labels. (right). The membrane tunnels for polysaccharide translocation are marked by the orange circles.